AbstractAcute myeloid leukemia is a neoplasm characterized by recurrent molecular aberrations traditionally demonstrated by cytogenetic analyses. We used high density genome-wide genotyping and gene expression profiling to reveal acquired cryptic abnormalities in acute myeloid leukemia. By genome-wide genotyping of 137 cases of primary acute myeloid leukemia, we disclosed a recurrent focal amplification on chromosome 14q32, which included the genes BCL11B, CCNK, C14orf177 and SETD3, in two cases. In the affected cases, the BCL11B gene showed consistently high mRNA expression, whereas the expression of the other genes was unperturbed. Fluorescence in situ hybridization on 40 cases of acute myeloid leukemia with high BCL11B mRNA expression [2.5-fold above median; 40 out of 530 cases (7.5%)] revealed 14q32 abnormalities in two additional cases. In the four BCL11B-rearranged cases the 14q32 locus was fused to different partner chromosomes. In fact, in two cases, we demonstrated that the focal 14q32 amplifications were integrated into transcriptionally active loci. The translocations involving BCL11B result in increased expression of full-length BCL11B protein. The BCL11B-rearranged acute myeloid leukemias expressed both myeloid and T-cell markers. These biphenotypic acute leukemias all carried FLT3 internal tandem duplications, a characteristic marker of acute myeloid leukemia. BCL11B mRNA expression in acute myeloid leukemia appeared to be strongly associated with expression of other T-cell-specific genes. Myeloid 32D(GCSF-R) cells ectopically expressing Bcl11b showed decreased proliferation rate and less maturation. In conclusion, by an integrated approach involving high-throughput genome-wide genotyping and gene expression profiling we identified BCL11B as a candidate oncogene in acute myeloid leukemia.
Acute myeloid leukemia (AML) is a heterogeneous clonal neoplasm characterized by accumulated genetic aberrations, which result in enhanced proliferation, blocked differentiation and increased survival of the leukemic blast cells and a variable response to therapy.21 In the past decades a number of recurrent cytogenetic abnormalities have been identified in AML, such as the chromosomal aberrations t(8;21) and inv(16).21 These recurrent molecular lesions result in expression of fusion proteins of which the leukemic potential, in combination with additional genetic events, has been demonstrated in both in vitro and in vivo models.3 In addition to cytogenetic abnormalities, acquired mutations in disease genes, such as FLT3, NPM1, RUNX1 and CEBPA, have recently been demonstrated to be involved in AML as well.21 Several acquired molecular aberrations carry prognostic value and have been incorporated in routine molecular analyses of AML.21
Nowadays, various genome-wide approaches, such as gene expression profiling, genome-wide genotyping and next-generation sequencing, enable detailed analyses of hematologic malignancies to identify novel pathogenic genes.542 Examples of aberrations in myeloid proliferative malignancies revealed with these novel technologies are mutations in the genes IDH1, TET2, DNMT3A, ASXL1, and EZH2.116
Besides balanced translocations, large chromosomal regions showing losses or gains of genetic material are apparent in the leukemic blasts of AML patients, e.g., those involving chromosome 5 and 7.1 In the past two decades, attempts to identify the tumor suppressor genes located on these chromosomes have failed. Through genome-wide single nucleotide polymorphism genotyping it has become possible to simultaneously genotype hundreds of thousands of single nucleotide polymorphisms in a single assay. In addition, single nucleotide polymorphism platforms can also be conveniently used to determine chromosomal copy numbers, similarly to array comparative genomic hybridization. Genomic DNA can be examined with an inter-marker distance of several hundreds of base pairs, which makes it feasible to detect (micro) deletions and/or amplifications that are missed with conventional cytogenetics. The application of high-throughput single nucleotide polymorphism genotyping has been elegantly demonstrated to be powerful for the identification of disease genes, such as those for acute lymphoblastic leukemia13124 or AML.14 Another major advantage of single nucleotide polymorphism arrays is the fact that allele losses are directly recognizable as loss-of-heterozygosity. In fact, single nucleotide polymorphism arrays revealed that approximately 20% of cases of AML exhibit large nonrandom regions of homozygosity without changes in copy number as a result of segmental uniparental disomy, often indicating mutations in genes within these regions. These areas of uniparental disomy have been associated with mutations in CEBPA, WT1, FLT3 and RUNX1.1615
In addition, deletion, amplification and uniparental disomy may have other important consequences, such as those on gene expression. Juxtaposition of regulatory sequences may result in increased or decreased expression of affected genes. Thus, genome-wide analyses to detect copy number changes and loss of heterozygosity in the context of gene expression may also point towards pathogenic genes. We recently developed SNPExpress, an easily accessible software tool to analyze single nucleotide polymorphism genotype calls, copy number and gene expression in an accurate, efficient combinatorial way.17
In this study, we identified BCL11B as the candidate oncogene in AML through an integrated approach of genome-wide genotyping and gene expression profiling, followed by next-generation sequencing. BCL11B is a Kruppel family zinc finger family gene located at 14q32, associated with transcriptional co-repressor complexes in mammalian cells and a pivotal regulator of differentiation and survival of hematopoietic cells, especially T cells.18 We demonstrated that BCL11B is involved in a number of cryptic 14q32 translocations in AML, in which BCL11B and T-cell associated genes expression levels are increased concomitantly. Overexpression of BCL11B in a murine myeloid cell line model inhibits proliferation.
This study was approved by our local Medical Ethical Committee (MEC-2004-030 and MEC2007-364). After informed consent, bone marrow aspirates or peripheral blood samples were collected from a representative cohort of AML patients. Eligible patients had a diagnosis of primary AML, confirmed by cytological examination of blood and bone marrow. All patients were treated according to HOVON (Dutch-Belgian Hematology-Oncology Co-operative group) protocols (http://www.hovon.nl). Further details are available in the Online Supplementary Appendix.
Genome-wide genotyping and gene expression profiling
Genome-wide genotyping data sets of 48 patients with various subtypes of AML were generated using Affymetrix 500K NspI/StyI DNA mapping arrays and of 89 patients with cytogenetically normal AML using Affymetrix 250K NspI or StyI DNA Mapping arrays. The copy numbers of all AML samples were calculated using diploid references, i.e., 15 normal karyotype AML samples. Further details are available in the Online Supplementary Appendix.
Gene expression profiles of the same AML cases were generated using Affymetrix HG-U133 plus 2.0, as described elsewhere (GEO Series accession number GSE6891).19 Pearson correlation analyses were performed as described previously.20
The genome-wide genotyping and gene expression profiling data sets were examined using SNPExpress.17
Fluorescence in situ hybridization
Dual color fluorescence in situ hybridization (FISH) was performed with BAC clones RP11-431B1, RP11-876E22, RP11-830F3, RP11-782I5, RP11-450C22, RP11-57E12, RP11-1069L3 and RP11-242A7 covering the BCL11B region and regions up- and downstream (BACPAC resources, Oakland, USA). Further details are available in the Online Supplementary Appendix.
Targeted sequencing of the 14q32 genomic region
Library preparation and targeted re-sequencing was performed following previously described protocols.21 In brief, high molecular weight DNA from AML #2301 and #7073 was sheared using a Covaris E210 waterbath sonificator. The BCL11B 14q32 - tel genomic region [chr14:93930247-105928955 (hg19)] was captured with a Roche/Nimblegen SeqCap EZ Choice XL Library. The captured region was subsequently paired-end sequenced using Illumina HiSeq2000. The data were analyzed using an in-house pipeline, including visualization with IGV, which identifies single nucleotide variants, small and large indels and copy number variations. The chromosomal breakpoints were identified using Breakdancer22 in the 14q32 region and the partner chromosome. The genomic fusions were subsequently confirmed by Sanger sequencing.
Western blot analyses
Western blot analyses were carried out using affinity-purified rabbit polyclonal anti-BCL11B antibody (Novus Biologicals, Littleton, USA). Immune complexes were detected by binding anti-mouse IgG conjugated to horseradish peroxidase (DAKO, Heverlee, Belgium) followed by an enhanced chemiluminescence assay (Amersham Bioscience, Piscataway, NJ, USA) and GAPDH was stained with primary affinity-purified rabbit polyclonal antibody (α-GAPDH FL-335) (Santa Cruz Biotechnology, California, USA). Further details are available in the Online Supplementary Appendix.
DNA constructs and generation of BCL11B-expressing 32D/GCSFR cells
A murine Bcl11b cDNA (kindly donated by Dorina Avram, Albany Medical Center, Albany, NY, USA) was subcloned into a pLXSN expression vector under the control of the 5′ long terminal repeat of the Moloney murine sarcoma virus (Clontech, Mountainview, USA). Vector constructs were confirmed by nucleotide sequencing and retrovirally transfected into 32D cells that stably express human granulocyte colony-stimulating factor receptor (GCSF-R)23 using Fugene transfection reagent (Roche, Indianapolis, USA). Cells were stimulated with interleukin-3 (25 ng/mL) or granulocyte colony-stimulating factor (25 ng/mL), counted and assessed for proliferation and granulocytic differentiation. Further details are available in the Online Supplementary Appendix.
Genome-wide genotyping of cytogenetically abnormal and normal cases of acute myeloid leukemia
In total, DNA mapping array profiles of 137 patients with AML were generated (Figure 1). Initially a subgroup of 48 AML samples was selected based on previous gene expression profiling studies, i.e., 21 AML cases from gene expression profiling clusters #4 and #15 (100% CEBPA mutant or CEBPA silenced24), 13 AML cases from cluster #9 [100% inv(16)] and 14 AML cases from cluster #10 (adverse prognosis).20 In addition, DNA mapping array profiles, i.e., Affymetrix 250K NspI or StyI DNA mapping array, of 89 AML cases with normal karyotypes were generated.
With the Affymetrix 500K NspI/StyI DNA Mapping arrays, all known numerical cytogenetic aberrations, i.e., whole chromosome and interstitial deletions and amplifications that had been identified with cytogenetic banding analysis, were recognized in the 48 cytogenetically abnormal AML samples, as long as the abnormalities were present in over 30% of the AML cells.17 Also, in approximately 25% of all cases large regions of segmental uniparental disomy were detected, often involving whole chromosome arms.17
In addition to the known cytogenetic aberrations, relatively low numbers of small interstitial deletions and amplifications were detected in the 137 AML cases. However, some of these were indicative of the presence of cryptic translocations, such as cryptic t(5;11), t(9;22) and t(4;11), which are known to encode chimeric fusion proteins essential for leukemogenesis. All fusion transcripts involved in these translocations, i.e., NUP98-NSD1,25 BCR-ABL and MLL-AF4, were confirmed by reverse transcriptase polymerase chain reaction analysis. Thus, although relatively small numbers of aberrations were found, most being non-recurrent, they may reliably tag relevant leukemic lesions.
Integrated analysis of genome-wide genotypes and gene expression profiles
By an integrated approach using the genome-wide genotyping data and previously determined gene expression profiles of the primary AML samples (Figure 1),20 we searched for genes aberrantly expressed as a result of numerical changes in the AML genome. Using SNPExpress,17 we identified two AML cases with relatively small interstitial amplifications in the 14q32 region (#2301 amplification: 482 Kb, 3 copies and #7073 amplification: 460 Kb, 3 copies) (Figure 2A,B). The amplified region encompassed the genes BCL11B, CCNK, C14orf177 and SETD3. Interestingly, BCL11B mRNA was highly expressed in the two AML cases with numerical changes, whereas the expression of C14orf177, CCNK and SETD3 were not affected as compared to other AML cases (Figure 2A,B). In addition, BCL11B mRNA was highly expressed in AML #2301 and #7073, whereas expression was low or absent in control AML cases (Figure 2A). This could indicate that BCL11B becomes overexpressed as a result of a genomic rearrangement in these AML cases. The small interstitial amplifications in AML #2301 and #7073 may point towards cryptic translocations, as was seen in AML cases carrying the cryptic translocations t(5;11), t(9;22) and t(4;11).
Fluorescence in situ hybridization reveals translocations in acute myeloid leukemia #2301 and #7073 involving BCL11B
To confirm the amplifications in the BCL11B locus in the two AML cases, we performed FISH analysis with a probe covering the BCL11B gene (RP11-431B1) and a probe flanking this locus (RP11-74H1) (Figure 3A). On metaphase spreads of both AML cases an additional BCL11B allele was apparent (Figure 3B). This is in line with the expected copy number change for the BCL11B locus (n=3) as shown with SNPExpress (Figure 2A,B). In fact, through verification using chromosomal paints we showed that BCL11B was translocated to chromosome 6 in AML case #2301 and chromosome 8 in AML #7073 (data not shown).
Amplified 14q32 genomic regions are integrated into transcriptionally active loci
We performed 14q32 capture sequencing on AML cases #2301 and #7073. Paired-end sequencing of the captured region on the Illumina HiSeq2000 in AML case #2301 and case #7073 revealed the partner chromosomal regions on chromosome 6 and 8, respectively. Paired-end reads spanning 14q32 and chr6q25.3 (chr6:156717480 and chr14:99110325; chr6:156587275 and chr14:99748893) were identified in AML case #2301 and reads spanning 14q32 and 8q24.21 (chr8:130485869 and chr14:99179210) in case #7073. These breakpoints were confirmed by polymerase chain reaction analysis of genomic DNA, followed by Sanger sequencing. The BCL11B encoding-amplified DNA integrated into two transcriptionally active regions on chromosomes 6 and 8, i.e., on 6q25.3 into an expressed sequence tag sequence CB984582 and on 8q24.21 into the large non-coding RNA gene Coiled-Coil Domain Containing 26 (CCDC26). Both polyA genes were transcriptionally active in the two cases of AML, and various other AML cases, and are subjected to mRNA splicing as demonstrated by RNA sequencing on an Illumina HiSeq2500 (data not shown), indicating that these RNA are expressed in myeloid cells. No fusion transcripts between BCL11B and RNA encoded by the partner chromosomes could be detected by RNAseq, suggesting that regulatory sequences on chr6q25.3 and 8q24.21 may activate the BCL11B gene in the BCL11B-rearranged AML cases.
Acute myeloid leukemia case #2301 expresses full-length BCL11B
The translocations involving BCL11B could result in increased expression of either full-length BCL11B or a fusion protein involving BCL11B. In fact, BCL11B mRNA expression in AML#2301 and AML#7073, were, respectively, 12- and 8-fold over mean BCL11B mRNA expression in 530 AML cases (219528_s_at; 22895_s_at; 224310_s_at).19 Next, we examined the expression profiles obtained with Affymetrix Human Exon 1.0 ST Array for AML case #2301. This analysis showed that in AML #2301 all four exons of BCL11B were expressed at similarly high levels (data not shown). The fact that exon 1 of BCL11B, containing the ATG start codon, was expressed suggested that full-length BCL11B is expressed rather than a fusion protein in which parts of BCL11B are involved.
Protein lysates of AML case #2301 were available for western blot analyses. With western blot analyses of whole, cytoplasmic and nuclear cell lysates of AML #2301 we assessed both the size and localization of the BCL11B protein. Immunodetection with BCL11B antibodies confirmed the expression of full-length BCL11B protein (Figure 4). Moreover, BCL11B expression appeared to be restricted to the nucleus. Of note, full-length BCL11B was also highly expressed in AML case #2238, an AML without any known aberration involving BCL11B.
Fluorescence in situ hybridization analyses of selected acute myeloid leukemia cases with high BCL11B mRNA expression reveals additional cases with BCL11B translocations
The fact that FISH analysis of AML cases showing high expression of BCL11B mRNA revealed translocations involving BCL11B raised the possibility that other AML cases with aberrantly high BCL11B expression would also harbor translocations involving BCL11B. Gene expression profiling of 530 AML cases19 showed variable expression of BCL11B mRNA in subsets of AML, including case #2301 and #7073 (Figure 5A). Interestingly, AML cases with high BCL11B expression appear to cluster in several distinct clusters (Figure 5A), suggesting that BCL11B overexpression is associated with distinct AML subtypes. We selected 40 AML cases with increased BCL11B mRNA expression, i.e., >2.5-fold above mean BCL11B expression in primary AML (Figure 5A), and performed FISH analysis on the BCL11B chromosomal region.
FISH analyses revealed two additional AML cases with a BCL11B translocation (AML #6366 and #6451) (Figure 5A,B). With specific chromosomal paints, we showed that in AML case #6451 the BCL11B locus was translocated to chromosome 7 (data not shown). Further FISH could not be carried out on AML #6366 due to a lack of material.
Immunophenotyping and molecular analyses of acute myeloid leukemia cases carrying BCL11B aberrations
Immunophenotyping the AML cases demonstrated that those with BCL11B translocations expressed not only myeloid markers, but also lymphoid markers such as CD2, CD3, and CD7 (Table 1). Cytoplasmic CD3 expression was present in case #2301, suggesting that it may better correspond to a T-acute lymphoblastic leukemia, but cytoplasmic CD3 was absent in the remaining cases. In fact, these AML cases appeared to have a biphenotypic signature, i.e., expressing (early) myeloid as well as T-cell-associated markers.
Well-known recurrent molecular abnormalities were searched for in the AML cases carrying BCL11B aberrations. These analyses demonstrated, with no exception, that these AML cases carried mutations in the FLT3 gene, i.e., FLT3 internal tandem duplications (ITD) in all cases and one case had a concurrent FLT3 mutation in the tyrosine kinase domain (TKD) (Table 1). We did not identify mutations in K-RAS, N-RAS, c-KIT, IDH1, IDH2, ASXL1 or CEBPA in the AML cases carrying a BCL11B abnormality. Case #6366 also carried a DNMT3A mutation (Table 1).
We analyzed the immunophenotype of the BCL11B non-rearranged cases with BCL11B overexpression and did not find a specific pattern of T-cell specific markers. Several cases did express CD7, but this aberrant marker is relatively frequently present in AML (present in approximately 30% of cases). Moreover, we were unable to demonstrate a significant association between BCL11B overexpression and FLT3-ITD or -TKD mutations in BCL11B non-rearranged cases of AML.
BCL11B is aberrantly expressed in acute myeloid leukemia and associated with the T-cell gene expression signature
To investigate whether other AML cases with elevated BCL11B mRNA expression show full-length BCL11B expression, we carried out western blot analyses on a number of AML cases with high BCL11B mRNA expression. All analyzed samples with high BCL11B mRNA showed full-length BCL11B protein expression at variable levels (Figure 6). Because of the lack of specimens, BCL11B protein expression analyses in non-rearranged BCL11B cases was limited to those shown in Figures 4 and 6.
To examine which genes are co-expressed with BCL11B in AML, we performed a Pearson correlation analysis using Affymetrix gene expression profiling data from 530 AML cases.19 BCL11B co-regulated probe sets were calculated across all AML patients. The top 50 BCL11B correlating probe sets are highly associated with T cells and T-cell development (Online Supplementary Table S1). In fact, the majority of BCL11B-associated genes are T-cell specific genes, with CD3, TRBV19, IL32, LCK, TCF7 and CD2 being just a few examples among many others (Online Supplementary Table S1).
Increased Bcl11b expression results in decreased proliferation of the myeloid cell line 32D(GCSF-R)
To investigate the effect of Bcl11b expression on proliferation and differentiation, immortalized myeloblast-like murine bone marrow cells stably expressing human GCSF-R [32D(GCSF-R)] were transfected with full-length murine Bcl11b cDNA. Three 32D(GCSF-R) clones expressing Bcl11b were selected and incubated for 10 days in the presence of interleukin-3 or granulocyte colony-stimulating factor. Western blot analyses demonstrated that BCL11B was expressed at all time points (Figure 7A). BCL11B-expressing 32D(GCSF-R) clones showed a consistent decreased proliferation rate when cultured in the presence of interleukin in comparison to 32D(GCSF-R) clones containing a control, empty vector (Figure 7B).
We also evaluated the granulocytic differentiation abilities of the same 32D(GCSF-R) clones upon stimulation with granulocyte colony-stimulating factor. Morphological analyses of cytospins did not show consistent maturation defects in the BCL11B-expressing 32D(GCSF-R) clones. However, less maturation towards granulocytes in 32D(GCSF-R) cells expressing BCL11B and more undifferentiated blast cells compared to cells with empty vector were present in some 32D(GCSF-R) BCL11B- expressing clones (Figure 7C). This effect was most apparent in 32D(GCSF-R) BCL11B clones #4 and #12, the 32D(GCSF-R) clones with the highest expression levels of BCL11B (Figure 7A).
Simultaneous analyses of genome-wide genotyping and copy number data with gene expression profiling enable the identification of pathogenic genes that, as a result of genomic imbalances, are aberrantly expressed. By an integrated approach involving genome-wide genotyping and gene expression profiling, we identified BCL11B as a novel oncogene in AML. Interstitial amplification of 14q32 was initially revealed in two AML cases (#2301 and #7073) by genome-wide genotyping, showing relatively small amplifications, including the BCL11B gene. Although other genes were affected by these numerical genetic changes, these 14q32 aberrations resulted in unique BCL11B mRNA and full-length BCL11B protein overexpression. By FISH of a selection of 40 AML cases with high BCL11B mRNA expression, we identified two additional AML cases bearing BCL11B translocations (AML #6451 and AML #6366).
The BCL11 family has two members, BCL11A and BCL11B.18 Bcl11a was identified as a common retroviral insertion site (Evi9) in murine myeloid leukemias and is required for normal B-cell development.26 Mice carrying biallelic inactivation of Bcl11b developed thymic lymphomas, indicating that loss-of-function mutations in Bcl11b contribute to mouse lymphomagenesis and, possibly, to human cancer development.27 BCL11B is a fourexon gene located on 14q32, encoding a Kruppel family zinc finger transcription factor and a key regulator of differentiation and survival of thymocytes.18
BCL11B was first associated with hematologic malignancies due to its recurrent involvement with the homeo -box transcription factor TLX3 in a relatively high percentage of cases of pediatric and adult T-cell acute lymphoblastic leukemia carrying the cryptic t(5;14)(q35;q32).2928 Less frequently, T-cell acute lymphoblastic leukemia samples with an inv(14)(q11.2q32.31) carry an in-frame transcript of BCL11B and the T-cell receptor gene segment TRDV1. These cases of acute lymphoblastic leukemia do not express wild-type BCL11B transcripts, suggesting that BCL11B disruption may contribute to T-cell malignancies in humans.30 Interestingly, a DNA copy number and sequencing analysis approach recently revealed monoallelic BCL11B deletions and missense mutations in 10–15% of cases of T-cell acute lymphoblastic leukemia.3231 Structural homology modeling showed that several of the BCL11B mutations disrupted the structure of zinc finger domains required for DNA binding.
A number of myeloid, mixed-lineage, and non-lymphocytic leukemias with 14q32 abnormalities have been reported, however, in these instances the affected genes were not identified.3733 The first evidence of BCL11B involvement in 14q32 translocations in AML was reported by Bezrookove et al.34 These authors reported one case of t(6;14)(q25~q26;q32) in an adult with AML and used bacterial artificial chromosomes to demonstrate the involvement of BCL11B in that case.34 Due to a lack of the patient’s material, the investigators could neither establish the deregulation of BCL11B nor identify the partner genomic locus.34 Of note, the breakpoint in this AML case appeared to be located upstream of the BCL11B gene. This resembles the AML cases described here and suggests that the breakpoints in AML are clustered upstream, whereas in acute lymphoblastic leukemia they are downstream, of BCL11B.
Specific chromosomal paints demonstrated that different partner chromosomes were involved in the AML cases with a BCL11B translocation. The fact that in two AML cases the BCL11B-containing amplified region integrated in transcriptionally active large non-coding RNA may suggest that different regulatory regions of large non-coding RNA are capable of activating the BCL11B oncogene in these cases of AML. The breakpoints in AML #2301 and #7073 are 10 kb and 600 kb away from the transcriptional start site of BCL11B. There is no obvious reason why BCL11B specifically, and not the other genes, would be activated. Interestingly, however, the rearranged BCL11B allele in both AML #2301 and AML #7073 is juxtaposed to recently described super-enhancers, which have been shown to act as key oncogenic drivers.3938 These putative super-enhancers are present in the cell line MOLM-1 and seem to be linked to ARID1B on chromosome 6 (#2301) and GSDMC on chromosome 8 (#7073) and may be responsible for increased BCL11B expression. The selective overexpression of BCL11B may give the cells a specific advantage, whereas the other genes located on 14q32 would not.
Interestingly, BCL11B protein appeared to be expressed in additional cases of primary AML that do not carry BCL11B translocations. In these AML cases other mutations may be present or BCL11B may be activated by other means.
The 14q32 region, including BCL11B, is subject to translocation in T-cell acute lymphoblastic leukemia and acute mixed lineage leukemia.373323 In fact, the involvement of 14q32 translocations and BCL11B in AML has been debated.40 However, the immunophenotyping and molecular analyses of the AML samples with BCL11B translocations described here showed that these leukemias have a biphenotypic immunophenotype, but also all carry a common AML-associated FLT3 ITD mutation. These leukemias do, therefore, share a characteristic genetic feature with AML.
BCL11B is expressed in T-lymphocytes and T-cell leukemias and is a pivotal regulator of number of genes related to T-cell proliferation and differentiation such as IL2, NF-kappaB, TCRβ and p21.4541 It was shown recently that the expression of BCL11B in T-cell lines resulted in markedly increased apoptosis resistance following treatment with radiomimetic drugs accompanied by a cell cycle delay caused by accumulation of cells at G1.46 We examined the consequences of Bcl11b overexpression on proliferation and differentiation in a mouse myeloid 32D(GCSF-R) cell line model. The 32D(GCSF-R) cells, expressing full-length murine Bcl11b cDNA, had a consistently decreased proliferation rate compared to cells expressing the empty vector or to the parental untransfected cells. Upon stimulation with granulocyte colony-stimulating factor, 32D(GCSF-R) cells overexpressing Bcl11b showed less maturation towards granulocytes compared to cells expressing empty vector, giving supporting evidence that BCL11B is partially blocking or delaying differentiation in 32D(GCSF-R) cells. The decreased proliferation rate in BCL11B-expressing cells suggests that a proliferative mutation, such as a FLT3-ITD, may indeed be required for full-leukemic transformation.
In conclusion, we have shown that BCL11B is involved in 14q32 translocations with different putative chromosomal partners in well-characterized AML cases using high-throughput genome-wide genotyping, cytogenetics and gene expression profiling. In these translocations, full-length BCL11B is highly expressed concomitantly with T-cell specific markers. We speculate that due to the translocations, BCL11B expression is influenced by active transcriptional elements on the partner chromosomes resulting in high BCL11B expression and consequently T-cell associated genes. The murine cell line 32D(GCSF-R) overexpressing BCL11B shows decreased proliferation and partial delayed differentiation, which provides evidence that BCL11B may have suppressive and disruptive effects on cell proliferation and differentiation of myeloid cells. Altogether, these analyses indicate that BCL11B is a putative oncogene in AML with and possibly without aberrations involving 14q32.
We are indebted to the participants of the HOVON clinical trials, and our colleagues from the stem cell transplantation and molecular diagnostics laboratories who provided, collected and analyzed AML cell samples, respectively.
- The online version of this article has a Supplementary Appendix.
- Funding This work was supported by grants from the Dutch Cancer Society (Koningin Wilhelmina Fonds) and performed within the framework of CTMM, the Center for Translational Molecular Medicine (Leukemia BioCHIP project; grant 030-102).
- Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.
- Received August 18, 2013.
- Accepted January 16, 2014.
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